With a foundation in physics and radiation biology, my academic journey has been dedicated to mastering the complex interactions between various forms of radiation and their applications across the breadth of imaging techniques. My expertise extends to the design and execution of primary and metastatic mouse-tumor models, instrumental in advancing early disease detection and therapeutic innovation. I am highly skilled in the nuanced field of in vivo contrast-enhanced molecular imaging, utilizing an array of imaging modalities, and in assisting image-guided radiation therapies. My early research was pivotal in creating contrast agents for molecular imaging probes, specifically targeting markers in endothelial cells of primary and metastatic tumors. In the recent phase of my career, I have turned my attention to ultra-high dose rate (FLASH) irradiation technology for preclinical therapy. Through comprehensive research and experimentation, I have been at the forefront of configuring linear accelerators for optimal FLASH radiation delivery, a technique that promises to revolutionize cancer treatment by minimizing harm to healthy tissue while effectively targeting tumor cells. My investigative work has not only illuminated the underlying radiobiological principles of the FLASH effect but has also been pivotal in developing and fine-tuning specific in vivo irradiation strategies. This includes the meticulous engineering of mouse shields and collimators for anatomy-specific radiation delivery and the design and fabrication of dosimetric phantoms and protocols that ensure precise and reliable dosimetry.

Current Role at Stanford

Currently, I serve as the Deputy Director of the Pre-clinical Radiotherapy core of the Radiation Oncology department, where I lead a team of Physics Assistants focused on developing dosimetric consensus across pre-clinical orthovoltage and MeV irradiation platforms. We implement clinical calibration and quality assurance principles in preclinical settings. The core provides equipment training to users, offers treatment planning support, and provides services for custom 3D-design, fabrication, and characterization of collimators and shielding for in vivo radiation treatments.

Concurrently, I have been a research scientist at the Loo laboratory. Our laboratory stands at the forefront of FLASH radiobiology research, providing the only pre-clinical FLASH electron platforms to over 10 laboratories spanning various disciplines, while our multi-institutional and international collaborations focus on introducing dosimetric harmonization across various FLASH electron platforms.

Honors & Awards

  • Invited presentation, Oxford Cancer Imaging Center Retreat (2013, 2014)
  • Invited presentation, Oxford Institute Metastasis Symposium (2014)
  • Poster Prize, Aegean Conferences - 3rd International Conference for Tumour Microenvironment and Cellular Stress (2014)
  • Recognition of excellent research, Aegean Conferences - 12th International Conference on Complement Therapeutics (2019)

Education & Certifications

  • Cert., UCLA, Medical Physics (2023)
  • DPhil, University of Oxford, Radiation Biology (2014)
  • MSc, University of Oxford, Radiation Biology (2010)
  • MSc, University of Liverpool, Radiometrics: Instrumentation and Modelling (2009)
  • BSc, University of Liverpool, Physics with Medical Applications (2008)
  • Diploma, IIEK Neapoleos, Greece, Electrical Engineering (2000)

All Publications

  • Multi-Institutional Audit of FLASH and Conventional Dosimetry with a 3D-Printed Anatomically Realistic Mouse Phantom. International journal of radiation oncology, biology, physics Ashraf, M. R., Melemenidis, S., Liu, K., Grilj, V., Jansen, J., Velasquez, B., Connell, L., Schulz, J. B., Bailat, C., Libed, A., Manjappa, R., Dutt, S., Soto, L., Lau, B., Garza, A., Larsen, W., Skinner, L., Yu, A. S., Surucu, M., Graves, E. E., Maxim, P. G., Kry, S. F., Vozenin, M. C., Schüler, E., Loo, B. W. 2024


    We conducted a multi-institutional dosimetric audit between FLASH and conventional dose rate (CONV) electron irradiations by using an anatomically realistic 3D-printed mouse phantom.A CT scan of a live mouse was used to create a 3D model of bony anatomy, lungs, and soft tissue. A dual-nozzle 3D printer was used to print the mouse phantom using acrylonitrile butadiene styrene (∼1.02 g/cm3) and polylactic acid (∼1.24 g/cm3) simultaneously to simulate soft tissue and bone densities, respectively. The lungs were printed separately using lightweight polylactic acid (∼0.64 g/cm3). Hounsfield units (HU), densities and print-to-print stability of the phantoms were assessed. Three institutions were each provided a phantom, and each institution performed two replicates of irradiations at selected anatomic regions. The average dose difference between FLASH and CONV dose distributions and deviation from the prescribed dose were measured with radiochromic film.Compared to the reference CT scan, CT scans of the phantom demonstrated mass density differences of 0.10 g/cm3 for bone, 0.12 g/cm3 for lung, and 0.03 g/cm3 for soft tissue regions. Differences in HU between phantoms were <10 HU for soft tissue and bone, with lung showing the most variation (54 HU), but with minimal impact on dose distribution (<0.5%). Mean differences between FLASH and CONV decreased from the first to the second replicate (4.3% to 1.2%), while differences from the prescribed dose decreased for both CONV (3.6% to 2.5%) and FLASH (6.4% to 2.7%). Total dose accuracy suggests consistent pulse dose and pulse number, though these were not specifically assessed. Positioning variability was observed, likely due to the absence of robust positioning aids or image guidance.This study marks the first dosimetric audit for FLASH using a non-homogeneous phantom, challenging conventional calibration practices reliant on homogeneous phantoms. The comparison protocol offers a framework for credentialing multi-institutional studies in FLASH preclinical research to enhance reproducibility of biological findings.

    View details for DOI 10.1016/j.ijrobp.2024.03.017

    View details for PubMedID 38493902

  • Exploring deep learning for estimating the isoeffective dose of FLASH irradiation from mouse intestinal histology images. International journal of radiation oncology, biology, physics Fu, J., Yang, Z., Melemenidis, S., Viswanathan, V., Dutt, S., Manjappa, R., Lau, B., Soto, L. A., Ashraf, R., Skinner, L., Yu, S. J., Surucu, M., Casey, K. M., Rankin, E. B., Graves, E., Lu, W., Loo, B. W., Gu, X. 2024


    Ultra-high dose rate (FLASH) irradiation has been reported to reduce normal tissue damage compared with conventional dose rate (CONV) irradiation without compromising tumor control. This proof-of-concept study aims to develop a deep learning (DL) approach to quantify the FLASH isoeffective dose (dose of CONV that would be required to produce the same effect as the given physical FLASH dose) with post-irradiation mouse intestinal histological images.84 healthy C57BL/6J female mice underwent 16 MeV electron CONV (0.12Gy/s; n=41) or FLASH (200Gy/s; n=43) single fraction whole abdominal irradiation. Physical dose ranged from 12 to 16Gy for FLASH and 11 to 15Gy for CONV in 1Gy increments. 4 days after irradiation, 9 jejunum cross-sections from each mouse were H&E stained and digitized for histological analysis. CONV dataset was randomly split into training (n=33) and testing (n=8) datasets. ResNet101-based DL models were retrained using the CONV training dataset to estimate the dose based on histological features. The classical manual crypt counting (CC) approach was implemented for model comparison. Cross-section-wise mean squared error (CS-MSE) was computed to evaluate the dose estimation accuracy of both approaches. The validated DL model was applied to the FLASH dataset to map the physical FLASH dose into the isoeffective dose.The DL model achieved a CS-MSE of 0.20Gy2 on the CONV testing dataset compared with 0.40Gy2 of the CC approach. Isoeffective doses estimated by the DL model for FLASH doses of 12, 13, 14, 15, and 16 Gy were 12.19±0.46, 12.54±0.37, 12.69±0.26, 12.84±0.26, and 13.03±0.28 Gy, respectively.Our proposed DL model achieved accurate CONV dose estimation. The DL model results indicate that in the physical dose range of 13 to 16 Gy, the biological dose response of small intestinal tissue to FLASH irradiation is represented by a lower isoeffective dose compared to the physical dose. Our DL approach can be a tool for studying isoeffective doses of other radiation dose modifying interventions.

    View details for DOI 10.1016/j.ijrobp.2023.12.032

    View details for PubMedID 38171387

  • Improving radiotherapy in immunosuppressive microenvironments by targeting complement receptor C5aR1. The Journal of clinical investigation Beach, C., MacLean, D., Majorova, D., Melemenidis, S., Nambiar, D. K., Kim, R. K., Valbuena, G. N., Guglietta, S., Krieg, C., Darvish-Damavandi, M., Suwa, T., Easton, A., Hillson, L. V., McCulloch, A. K., McMahon, R. K., Pennel, K., Edwards, J., O'Cathail, S. M., Roxburgh, C. S., Domingo, E., Moon, E. J., Jiang, D., Jiang, Y., Zhang, Q., Koong, A. C., Woodruff, T. M., Graves, E. E., Maughan, T., Buczacki, S. J., Stucki, M., Le, Q. T., Leedham, S. J., Giaccia, A. J., Olcina, M. M. 2023


    An immunosuppressive microenvironment causes poor tumour T-cell infiltration and is associated with reduced patient overall survival in colorectal cancer. How to improve treatment responses in these tumours is still a challenge. Using an integrated screening approach to identify cancer-specific vulnerabilities, we identified complement receptor C5aR1 as a druggable target which when inhibited improved radiotherapy even in tumours displaying immunosuppressive features and poor CD8+ T-cell infiltration. While C5aR1 is well-known for its role in the immune compartment, we found that C5aR1 is also robustly expressed on malignant epithelial cells, highlighting potential tumour-cell specific functions. C5aR1 targeting resulted in increased NF-kB-dependent apoptosis specifically in tumours and not normal tissues; indicating that in malignant cells, C5aR1 primarily regulated cell fate. Collectively, these data revealed that increased complement gene expression is part of the stress response mounted by irradiated tumours and that targeting C5aR1 could improve radiotherapy even in tumours displaying immunosuppressive features.

    View details for DOI 10.1172/JCI168277

    View details for PubMedID 37824211

  • FLASH-RT does not affect chromosome translocations and junction structures beyond that of CONV-RT dose-rates. Radiotherapy and oncology : journal of the European Society for Therapeutic Radiology and Oncology Barghouth, P. G., Melemenidis, S., Montay-Gruel, P., Ollivier, J., Viswanathan, V., Jorge, P. G., Soto, L. A., Lau, B. C., Sadeghi, C., Edlabadkar, A., Zhang, R., Ru, N., Baulch, J. E., Manjappa, R., Wang, J., Le Bouteiller, M., Surucu, M., Yu, A., Bush, K., Skinner, L., Maxim, P. G., Loo, B. W., Limoli, C. L., Vozenin, M. C., Frock, R. L. 2023: 109906


    The impact of radiotherapy (RT) at ultra high vs conventional dose rate (FLASH vs CONV) on the generation and repair of DNA double strand breaks (DSBs) is an important question that remains to be investigated. Here, we tested the hypothesis as to whether FLASH-RT generates decreased chromosomal translocations compared to CONV-RT.We used two FLASH validated electron beams and high-throughput rejoin and genome-wide translocation sequencing (HTGTS-JoinT-seq), employing S. aureus and S. pyogenes Cas9 "bait" DNA double strand breaks (DSBs) in HEK239T cells, to measure differences in bait-proximal repair and their genome-wide translocations to "prey" DSBs generated after various irradiation doses, dose rates and oxygen tensions (normoxic, 21% O2; physiological, 4% O2; hypoxic, 2% and 0.5% O2). Electron irradiation was delivered using a FLASH capable Varian Trilogy and the eRT6/Oriatron at CONV (0.08-0.13Gy/s) and FLASH (1x102-5x106 Gy/s) dose rates. Related experiments using clonogenic survival and γH2AX foci in the 293T and the U87 glioblastoma lines were also performed to discern FLASH-RT vs CONV-RT DSB effects.Normoxic and physioxic irradiation of HEK293T cells increased translocations at the cost of decreasing bait-proximal repair but were indistinguishable between CONV-RT and FLASH-RT. Although no apparent increase in chromosome translocations was observed with hypoxia-induced apoptosis, the combined decrease in oxygen tension with IR dose-rate modulation did not reveal significant differences in the level of translocations nor in their junction structures. Furthermore, RT dose rate modality on U87 cells did not change γH2AX foci numbers at 1- and 24-hours post-irradiation nor did this affect 293T clonogenic survival.Irrespective of oxygen tension, FLASH-RT produces translocations and junction structures at levels and proportions that are indistinguishable from CONV-RT.

    View details for DOI 10.1016/j.radonc.2023.109906

    View details for PubMedID 37690668

  • Clinical LINAC-based electron FLASH: Pathway for practical translation to FLASH clinical trials: LINAC electron FLASH. International journal of radiation oncology, biology, physics No, H. J., Wu, Y. F., Dworkin, M. L., Manjappa, R., Skinner, L., Ashraf, M. R., Lau, B., Melemenidis, S., Viswanathan, V., Yu, A. S., Surucu, M., Schüler, E., Graves, E. E., Maxim, P. G., Loo, B. W. 2023


    Ultra-high dose rate (UHDR) radiotherapy (RT) has produced the FLASH effect in preclinical models: reduced toxicity with comparable tumor control compared to conventional dose rate RT. Early clinical trials focused on UHDR RT feasibility using specialized devices. We explore the technical feasibility of practical electron UHDR RT on a standard clinical linear accelerator (LINAC).We tuned the program board of a decommissioned electron energy for UHDR electron delivery on a clinical LINAC, without hardware modification. Pulse delivery was controlled using the respiratory gating interface. A short SSD electron set-up with a standard scattering foil was configured and tested on an anthropomorphic phantom using circular blocks with 3-20 cm field sizes. Dosimetry was evaluated using radiochromic film and an ion chamber profiler.UHDR open field mean dose rates at 100, 80, 70, and 59 cm SSD were 36.82, 59.52, 82.01, and 112.83 Gy/s, respectively. At 80 cm SSD, mean dose rate was ∼60 Gy/s for all collimated field sizes, with an R80 depth of 6.1 cm corresponding to an energy of 17.5 MeV. Heterogeneity was <5.0% with asymmetry of 2.2 to 6.2%. The short SSD set-up was feasible under realistic treatment conditions simulating broad clinical indications on an anthropomorphic phantom.Short SSD and tuning for high electron beam current on a standard clinical LINAC can deliver flat, homogenous UHDR electrons over a broad, clinically relevant range of field sizes and depths with practical working distances, in a configuration easily reversible to standard clinical use.

    View details for DOI 10.1016/j.ijrobp.2023.04.011

    View details for PubMedID 37105403

  • Human enteroids as a tool to study conventional and ultra-high dose rate radiation. Integrative biology : quantitative biosciences from nano to macro Klett, K. C., Martin-Villa, B. C., Villarreal, V. S., Melemenidis, S., Viswanathan, V., Manjappa, R., Ashraf, M. R., Soto, L., Lau, B., Dutt, S., Rankin, E. B., Loo, B. W., Heilshorn, S. C. 2023; 15


    Radiation therapy, one of the most effective therapies to treat cancer, is highly toxic to healthy tissue. The delivery of radiation at ultra-high dose rates, FLASH radiation therapy (FLASH), has been shown to maintain therapeutic anti-tumor efficacy while sparing normal tissues compared to conventional dose rate irradiation (CONV). Though promising, these studies have been limited mainly to murine models. Here, we leveraged enteroids, three-dimensional cell clusters that mimic the intestine, to study human-specific tissue response to radiation. We observed enteroids have a greater colony growth potential following FLASH compared with CONV. In addition, the enteroids that reformed following FLASH more frequently exhibited proper intestinal polarity. While we did not observe differences in enteroid damage across groups, we did see distinct transcriptomic changes. Specifically, the FLASH enteroids upregulated the expression of genes associated with the WNT-family, cell-cell adhesion, and hypoxia response. These studies validate human enteroids as a model to investigate FLASH and provide further evidence supporting clinical study of this therapy. Insight Box Promising work has been done to demonstrate the potential of ultra-high dose rate radiation (FLASH) to ablate cancerous tissue, while preserving healthy tissue. While encouraging, these findings have been primarily observed using pre-clinical murine and traditional two-dimensional cell culture. This study validates the use of human enteroids as a tool to investigate human-specific tissue response to FLASH. Specifically, the work described demonstrates the ability of enteroids to recapitulate previous in vivo findings, while also providing a lens through which to probe cellular and molecular-level responses to FLASH. The human enteroids described herein offer a powerful model that can be used to probe the underlying mechanisms of FLASH in future studies.

    View details for DOI 10.1093/intbio/zyad013

    View details for PubMedID 37874173

  • FLASH-RT does not affect chromosome translocations and junction structures beyond that of CONV-RT dose-rates. bioRxiv : the preprint server for biology Barghouth, P. G., Melemenidis, S., Montay-Gruel, P., Ollivier, J., Viswanathan, V., Jorge, P. G., Soto, L. A., Lau, B. C., Sadeghi, C., Edlabadkar, A., Manjappa, R., Wang, J., Bouteiller, M. L., Surucu, M., Yu, A., Bush, K., Skinner, L., Maxim, P. G., Loo, B. W., Limoli, C. L., Vozenin, M., Frock, R. L. 2023


    The molecular and cellular mechanisms driving the enhanced therapeutic ratio of ultra-high dose-rate radiotherapy (FLASH-RT) over slower conventional (CONV-RT) radiotherapy dose-rate remain to be elucidated. However, attenuated DNA damage and transient oxygen depletion are among several proposed models. Here, we tested whether FLASH-RT under physioxic (4% O 2 ) and hypoxic conditions (≤2% O 2 ) reduces genome-wide translocations relative to CONV-RT and whether any differences identified revert under normoxic (21% O 2 ) conditions. We employed high-throughput rejoin and genome-wide translocation sequencing ( HTGTS-JoinT-seq ), using S. aureus and S. pyogenes Cas9 "bait" DNA double strand breaks (DSBs), to measure differences in bait-proximal repair and their genome-wide translocations to "prey" DSBs generated by electron beam CONV-RT (0.08-0.13Gy/s) and FLASH-RT (1*10 2 -5*10 6 Gy/s), under varying ionizing radiation (IR) doses and oxygen tensions. Normoxic and physioxic irradiation of HEK293T cells increased translocations at the cost of decreasing bait-proximal repair but were indistinguishable between CONV-RT and FLASH-RT. Although no apparent increase in chromosome translocations was observed with hypoxia-induced apoptosis, the combined decrease in oxygen tension with IR dose-rate modulation did not reveal significant differences in the level of translocations nor in their junction structures. Thus, Irrespective of oxygen tension, FLASH-RT produces translocations and junction structures at levels and proportions that are indistinguishable from CONV-RT.

    View details for DOI 10.1101/2023.03.27.534408

    View details for PubMedID 37034651

  • Design and validation of a dosimetric comparison scheme tailored for ultra-high dose-rate electron beams to support multicenter FLASH preclinical studies. Radiotherapy and oncology : journal of the European Society for Therapeutic Radiology and Oncology Gonçalves Jorge, P., Melemenidis, S., Grilj, V., Buchillier, T., Manjappa, R., Viswanathan, V., Gondré, M., Vozenin, M. C., Germond, J. F., Bochud, F., Moeckli, R., Limoli, C., Skinner, L., Joshua No, H., Fred Wu, Y., Surucu, M., Yu, A. S., Lau, B., Wang, J., Schüler, E., Bush, K., Graves, E. E., Maxim, P. G., Loo, B. W., Bailat, C. 2022


    We describe a multicenter cross validation of ultra-high dose rate (UHDR) (>= 40 Gy/s) irradiation in order to bring a dosimetric consensus in absorbed dose to water. UHDR refers to dose rates over 100-1000 times those of conventional clinical beams. UHDR irradiations have been a topic of intense investigation as they have been reported to induce the FLASH effect in which normal tissues exhibit reduced toxicity relative to conventional dose rates. The need to establish optimal beam parameters capable of achieving the in vivo FLASH effect has become paramount. It is therefore necessary to validate and replicate dosimetry across multiple sites conducting UHDR studies with distinct beam configurations and experimental set-ups.Using a custom cuboid phantom with a cylindrical cavity (5 mm diameter by 10.4 mm length) designed to contain three type of dosimeters (thermoluminescent dosimeters (TLDs), alanine pellets, and Gafchromic films), irradiations were conducted at expected doses of 7.5 to 16 Gy delivered at UHDR or conventional dose rates using various electron beams at the Radiation Oncology Departments of the CHUV in Lausanne, Switzerland and Stanford University, CA.Data obtained between replicate experiments for all dosimeters were in excellent agreement (+/- 3 %). In general, films and TLDs were in closer agreement with each other, while alanine provided the closest match between the expected and measured dose, with certain caveats related to absolute reference dose.In conclusion, successful cross-validation of different electron beams operating under different energies and configurations lays the foundation for establishing dosimetric consensus for UHDR irradiation studies, and, if widely implemented, decrease uncertainty between different sites investigating the mechanistic basis of the FLASH effect.

    View details for DOI 10.1016/j.radonc.2022.08.023

    View details for PubMedID 36030934

  • Real-time optical oximetry during FLASH radiotherapy using a phosphorescent nanoprobe. Radiotherapy and oncology : journal of the European Society for Therapeutic Radiology and Oncology Ha, B., Liang, K., Liu, C., Melemenidis, S., Manjappa, R., Viswanathan, V., Das, N., Ashraf, R., Lau, B., Soto, L., Graves, E. E., Rao, J., Loo, B. W., Pratx, G. 2022


    The rapid depletion of oxygen during irradiation at ultra-high dose rate calls for tissue oximeters capable of high temporal resolution. This study demonstrates a water-soluble phosphorescent nanoprobe and fiber-coupled instrument, which together are used to measure the kinetics of oxygen depletion at 200 Hz during irradiation of in vitro solutions.

    View details for DOI 10.1016/j.radonc.2022.08.011

    View details for PubMedID 35964762

  • Endogenous Retroviral Elements Generate Pathologic Neutrophils in Pulmonary Arterial Hypertension. American journal of respiratory and critical care medicine Taylor, S., Isobe, S., Cao, A., Contrepois, K., Benayoun, B. A., Jiang, L., Wang, L., Melemenidis, S., Ozen, M. O., Otsuki, S., Shinohara, T., Sweatt, A. J., Kaplan, J., Moonen, J., Marciano, D. P., Gu, M., Miyagawa, K., Hayes, B., Sierra, R. G., Kupitz, C. J., Del Rosario, P. A., Hsi, A., Thompson, A. A., Ariza, M. E., Demirci, U., Zamanian, R. T., Haddad, F., Nicolls, M. R., Snyder, M. P., Rabinovitch, M. 2022


    RATIONALE: The role of neutrophils and their extracellular vesicles (EVs) in the pathogenesis of pulmonary arterial hypertension is unclear.OBJECTIVES: Relate functional abnormalities in pulmonary arterial hypertension neutrophils and their EVs to mechanisms uncovered by proteomic and transcriptomic profiling.METHODS: Production of elastase, release of extracellular traps, adhesion and migration were assessed in neutrophils from pulmonary arterial hypertension patients and control subjects. Proteomic analyses were applied to explain functional perturbations, and transcriptomic data were used to find underlying mechanisms. CD66b-specific neutrophil EVs were isolated from plasma of patients with pulmonary arterial hypertension and we determined whether they produce pulmonary hypertension in mice.MEASUREMENTS AND MAIN RESULTS: Neutrophils from pulmonary arterial hypertension patients produce and release increased neutrophil elastase, associated with enhanced extracellular traps. They exhibit reduced migration and increased adhesion attributed to elevated beta1integrin and vinculin identified on proteomic analysis and previously linked to an antiviral response. This was substantiated by a transcriptomic interferon signature that we related to an increase in human endogenous retrovirus k envelope protein. Transfection of human endogenous retrovirus k envelope in a neutrophil cell line (HL-60) increases neutrophil elastase and interferon genes, whereas vinculin is increased by human endogenous retrovirus k dUTPase that is elevated in patient plasma. Neutrophil EVs from patient plasma contain increased neutrophil elastase and human endogenous retrovirus k envelope and induce pulmonary hypertension in mice, mitigated by elafin, an elastase inhibitor.CONCLUSIONS: Elevated human endogenous retroviral elements and elastase link a neutrophil innate immune response to pulmonary arterial hypertension.

    View details for DOI 10.1164/rccm.202102-0446OC

    View details for PubMedID 35696338

  • Abdominopelvic FLASH Irradiation Improves PD-1 Immune Checkpoint Inhibition in Preclinical Models of Ovarian Cancer. Molecular cancer therapeutics Eggold, J. T., Chow, S., Melemenidis, S., Wang, J., Natarajan, S., Loo, P. E., Manjappa, R., Viswanathan, V., Kidd, E. A., Engleman, E., Dorigo, O., Loo, B. W., Rankin, E. B. 2021


    Treatment of advanced ovarian cancer using PD-1/PD-L1 immune checkpoint blockade shows promise, however current clinical trials are limited by modest response rates. Radiation therapy has been shown to synergize with PD-1/PD-L1 blockade in some cancers but has not been utilized in advanced ovarian cancer due to toxicity associated with conventional abdominopelvic irradiation. While ultra-high dose rate (FLASH) irradiation has emerged as a strategy to reduce radiation-induced toxicity, the immunomodulatory properties of FLASH irradiation remain unknown. Here we demonstrate that single high dose abdominopelvic FLASH irradiation promoted intestinal regeneration and maintained tumor control in a preclinical mouse model of ovarian cancer. Reduced tumor burden in conventional and FLASH treated mice was associated with an early decrease in intratumoral regulatory T cells and a late increase in cytolytic CD8+ T cells. Compared to conventional irradiation, FLASH irradiation increased intratumoral T cell infiltration at early timepoints. Moreover, FLASH irradiation maintained the ability to increase intratumoral CD8+ T cell infiltration and enhance the efficacy of alphaPD-1 therapy in preclinical models of ovarian cancer. These data highlight the potential for FLASH irradiation to improve the therapeutic efficacy of checkpoint inhibition in the treatment of ovarian cancer.

    View details for DOI 10.1158/1535-7163.MCT-21-0358

    View details for PubMedID 34866044

  • Multicellular spheroids as in vitro models of oxygen depletion during FLASH irradiation. International journal of radiation oncology, biology, physics Khan, S., Bassenne, M., Wang, J., Manjappa, R., Melemenidis, S., Breitkreutz, D. Y., Maxim, P. G., Xing, L., Loo, B. W., Pratx, G. 2021


    PURPOSE: The differential response of normal and tumor tissues to ultra-high dose rate radiation (FLASH) has raised new hope for treating solid tumors but, to date, the mechanism remains elusive. One leading hypothesis is that FLASH radiochemically depletes oxygen from irradiated tissues faster than it is replenished through diffusion. The purpose of this study is to investigate these effects within hypoxic multicellular tumor spheroids, through simulations and experiments.MATERIALS AND METHODS: Physicobiological equations were derived to model (i) the diffusion and metabolism of oxygen within spheroids; (ii) its depletion through reactions involving radiation-induced radicals; and (iii) the increase in radioresistance of spheroids, modeled according to the classical oxygen enhancement ratio and linear-quadratic response. These predictions were then tested experimentally in A549 spheroids exposed to electron irradiation at conventional (0.075 Gy/s) or FLASH (90 Gy/s) dose rates. Clonogenic survival, cell viability, and spheroid growth were scored post-radiation. Clonogenic survival of two other cell lines was also investigated.RESULTS: The existence of a hypoxic core in unirradiated tumor spheroids is predicted by simulations and visualized by fluorescence microscopy. Upon FLASH irradiation, this hypoxic core transiently expands, engulfing a large number of well-oxygenated cells. In contrast, oxygen is steadily replenished during slower conventional irradiation. Experimentally, clonogenic survival was around 3-fold higher in FLASH-irradiated spheroid compared to conventional irradiation, but no significant difference was observed for well-oxygenated 2D-cultured cells. This differential survival is consistent with the predictions of the computational model. FLASH irradiation of spheroids resulted in a dose-modifying factor of around 1.3 for doses above 10 Gy.CONCLUSION: Tumor spheroids can be used as a model to study FLASH irradiation in vitro . The improved survival of tumor spheroids receiving FLASH radiation confirms that ultra-fast radiochemical oxygen depletion and its slow replenishment are critical components of the FLASH effect.

    View details for DOI 10.1016/j.ijrobp.2021.01.050

    View details for PubMedID 33545301

  • Reprogramming of serine metabolism during breast cancer progression Li, A., Ducker, G. S., Li, Y., Seoane, J. A., Xiao, Y., Melemenidis, S., Zhou, Y., Liu, L., Vanharanta, S., Graves, E. E., Rankin, E. B., Curtis, C., Massague, J., Rabinowitz, J. D., Thompson, C. B., Ye, J. AMER ASSOC CANCER RESEARCH. 2020
  • Increased local tumor control through nanoparticle-mediated, radiation-triggered release of nitrite, an important precursor for reactive nitrogen species. Physics in medicine and biology Kim, A. S., Melemenidis, S., Gustavsson, A. K., Abid, D., Wu, Y., Liu, F., Hristov, D., Schuler, E. 2020


    The efficacy of dose-enhancing gold nanoparticles (AuNPs) is negatively impacted by low tumor uptake, low cell membrane penetration, limited diffusion distance, and short lifetime of radiation-induced secondary particles. To overcome these limitations, we have developed a novel AuNP system capable of radiation-triggered release of nitrite, a precursor of reactive nitrogen species (RNS), and report here on the in vivo characterization of this system. AuNPs were functionalized through PEGylation, cell-penetrating peptides (CPP; AuNP@CPP), and nitroimidazole (nIm; AuNP@nIm-CPP). Mice with subcutaneous 4T1 tumors received either AuNP@nIm-CPP or AuNP@CPP intraperitoneally. Tumor and normal tissue uptake were evaluated 24 hours post AuNP administration. A separate cohort of mice was injected and irradiated to a single-fraction dose of 18Gy in a 225 kVp small animal irradiator 24 hours post NP administration. The mice were followed for two weeks to evaluate tumor response. The mean physical and hydrodynamic size of both NP systems were 5nm and 13nm, respectively. NP nIm-loading of 1wt% was determined. Tumor accumulation of AuNP@nIm-CPP was significantly lower than that of AuNP@CPP (0.2% vs 1.2%, respectively). In contrast, AuNP@nIm-CPP showed higher accumulation compared to AuNP@CPP in liver (16.5% vs 6.6%, respectively) and spleen (10.8% vs 3.1%, respectively). With respect to tumor response, no differential response was found between non-irradiated mice receiving either saline or AuNP@nIm-CPP alone. The combination of AuNP@CPP+radiation showed no differential response from radiation alone. In contrast, a significant delay in tumor regrowth was observed in mice receiving AuNP@nIm-CPP+radiation compared to radiation alone. AuNP functionalized with both CPP and nIm exhibited an order of magnitude less tumor accumulation compared to the NP system without nIm yet resulted in a significantly higher therapeutic response. Our data suggest that by improving the biokinetics of AuNP@nIm-CPP, this novel NP system could be a promising radiosensitizer for enhanced therapeutic response following radiation therapy.

    View details for DOI 10.1088/1361-6560/abaa27

    View details for PubMedID 32721936

  • Abdominal FLASH irradiation reduces radiation-induced gastrointestinal toxicity for the treatment of ovarian cancer in mice. Scientific reports Levy, K. n., Natarajan, S. n., Wang, J. n., Chow, S. n., Eggold, J. T., Loo, P. E., Manjappa, R. n., Melemenidis, S. n., Lartey, F. M., Schüler, E. n., Skinner, L. n., Rafat, M. n., Ko, R. n., Kim, A. n., H Al-Rawi, D. n., von Eyben, R. n., Dorigo, O. n., Casey, K. M., Graves, E. E., Bush, K. n., Yu, A. S., Koong, A. C., Maxim, P. G., Loo, B. W., Rankin, E. B. 2020; 10 (1): 21600


    Radiation therapy is the most effective cytotoxic therapy for localized tumors. However, normal tissue toxicity limits the radiation dose and the curative potential of radiation therapy when treating larger target volumes. In particular, the highly radiosensitive intestine limits the use of radiation for patients with intra-abdominal tumors. In metastatic ovarian cancer, total abdominal irradiation (TAI) was used as an effective postsurgical adjuvant therapy in the management of abdominal metastases. However, TAI fell out of favor due to high toxicity of the intestine. Here we utilized an innovative preclinical irradiation platform to compare the safety and efficacy of TAI ultra-high dose rate FLASH irradiation to conventional dose rate (CONV) irradiation in mice. We demonstrate that single high dose TAI-FLASH produced less mortality from gastrointestinal syndrome, spared gut function and epithelial integrity, and spared cell death in crypt base columnar cells compared to TAI-CONV irradiation. Importantly, TAI-FLASH and TAI-CONV irradiation had similar efficacy in reducing tumor burden while improving intestinal function in a preclinical model of ovarian cancer metastasis. These findings suggest that FLASH irradiation may be an effective strategy to enhance the therapeutic index of abdominal radiotherapy, with potential application to metastatic ovarian cancer.

    View details for DOI 10.1038/s41598-020-78017-7

    View details for PubMedID 33303827

  • FLASH Irradiation Results in Reduced Severe Skin Toxicity Compared to Conventional-Dose-Rate Irradiation. Radiation research Soto, L. A., Casey, K. M., Wang, J. n., Blaney, A. n., Manjappa, R. n., Breitkreutz, D. n., Skinner, L. n., Dutt, S. n., Ko, R. B., Bush, K. n., Yu, A. S., Melemenidis, S. n., Strober, S. n., Englemann, E. n., Maxim, P. G., Graves, E. E., Loo, B. W. 2020


    Radiation therapy, along with surgery and chemotherapy, is one of the main treatments for cancer. While radiotherapy is highly effective in the treatment of localized tumors, its main limitation is its toxicity to normal tissue. Previous preclinical studies have reported that ultra-high dose-rate (FLASH) irradiation results in reduced toxicity to normal tissues while controlling tumor growth to a similar extent relative to conventional-dose-rate (CONV) irradiation. To our knowledge this is the first report of a dose-response study in mice comparing the effect of FLASH irradiation vs. CONV irradiation on skin toxicity. We found that FLASH irradiation results in both a lower incidence and lower severity of skin ulceration than CONV irradiation 8 weeks after single-fraction hemithoracic irradiation at high doses (30 and 40 Gy). Survival was also higher after FLASH hemithoracic irradiation (median survival >180 days at doses of 30 and 40 Gy) compared to CONV irradiation (median survival 100 and 52 days at 30 and 40 Gy, respectively). No ulceration was observed at doses 20 Gy or below in either FLASH or CONV. These results suggest a shifting of the dose-response curve for radiation-induced skin ulceration to the right for FLASH, compared to CONV irradiation, suggesting the potential for an enhanced therapeutic index for radiation therapy of cancer.

    View details for DOI 10.1667/RADE-20-00090

    View details for PubMedID 32853385

  • Evaluating the Reproducibility of Mouse Anatomy under Rotation in a Custom Immobilization Device for Conformal FLASH Radiotherapy. Radiation research Ko, R. B., Soto, L. A., von Eyben, R. n., Melemenidis, S. n., Rankin, E. B., Maxim, P. G., Graves, E. E., Loo, B. W. 2020


    The observation of an enhanced therapeutic index for FLASH radiotherapy in mice has created interest in practical laboratory-based FLASH irradiators. To date, systems capable of 3D conformal FLASH irradiation in mice have been lacking. We are developing such a system, incorporating a high-current linear accelerator to produce a collimated X-ray beam in a stationary beamline design, rotating the mouse about a longitudinal axis to achieve conformal irradiation from multiple beam directions. The purpose of this work was to evaluate the reproducibility of mouse anatomy under rotation at speeds compatible with conformal FLASH delivery. Three short-hair mice and two hairless mice were immobilized under anesthesia in body weight-specific contoured plastic molds, and subjected to three rotational (up to 3 revolutions/s) and two non-rotational movement interventions. MicroCT images were acquired before and after each intervention. The displacements of 11 anatomic landmarks were measured on the image pairs. The displacement of the anatomical landmarks with any of the interventions was 0.5 mm or less for 92.4% of measurements, with a single measurement out of 275 (11 landmarks × 5 interventions × 5 mice) reaching 1 mm. There was no significant difference in the displacements associated with rotation compared to those associated with moving the immobilized mouse in and out of a scanner or with leaving the mouse in place for 5 min with no motion. There were no significant differences in displacements between mice with or without hair, although the analysis is limited by small numbers, or between different anatomic landmarks. These results show that anatomic reproducibility under rotation speed corresponding to FLASH irradiation times appears to be compatible with conformal/stereotactic irradiation in mice.

    View details for DOI 10.1667/RADE-20-00095

    View details for PubMedID 32857849

  • Metabolic Profiling Reveals a Dependency of Human Metastatic Breast Cancer on Mitochondrial Serine and One-Carbon Unit Metabolism. Molecular cancer research : MCR Li, A. M., Ducker, G. S., Li, Y. n., Seoane, J. A., Xiao, Y. n., Melemenidis, S. n., Zhou, Y. n., Liu, L. n., Vanharanta, S. n., Graves, E. E., Rankin, E. B., Curtis, C. n., Massague, J. n., Rabinowitz, J. D., Thompson, C. B., Ye, J. n. 2020


    Breast cancer is the most common cancer among American women and a major cause of mortality. To identify metabolic pathways as potential targets to treat metastatic breast cancer, we performed metabolomics profiling on breast cancer cell line MDA-MB-231 and its tissue-tropic metastatic subclones. Here, we report that these subclones with increased metastatic potential display an altered metabolic profile compared to the parental population. In particular, the mitochondrial serine and one-carbon (1C) unit pathway is upregulated in metastatic subclones. Mechanistically, the mitochondrial serine and 1C unit pathway drives the faster proliferation of subclones through enhanced de novo purine biosynthesis. Inhibition of the first rate-limiting enzyme of the mitochondrial serine and 1C unit pathway, serine hydroxymethyltransferase (SHMT2), potently suppresses proliferation of metastatic subclones in culture and impairs growth of lung metastatic subclones at both primary and metastatic sites in mice. Some human breast cancers exhibit a significant association between the expression of genes in the mitochondrial serine and 1C unit pathway with disease outcome and higher expression of SHMT2 in metastatic tumor tissue compared to primary tumors. In addition to breast cancer, a few other cancer types, such as adrenocortical carcinoma (ACC) and kidney chromophobe cell carcinoma (KICH), also display increased SHMT2 expression during disease progression. Together, these results suggest that mitochondrial serine and 1C unit plays an important role in promoting cancer progression, particularly in late stage cancer. Implications: This study identifies mitochondrial serine and 1C unit metabolism as an important pathway during the progression of a subset of human breast cancers.

    View details for DOI 10.1158/1541-7786.MCR-19-0606

    View details for PubMedID 31941752

  • Theranostic nanoparticles enhance the response of glioblastomas to radiation Nanotheranostics Wu, W., Klockow, J. L., Mohanty, S., Ku, K. S., Daldrup-Link, H. E. 2019; 3(4) (299-310)

    View details for DOI 10.7150/ntno.35342

  • The tumour microenvironment links complement system dysregulation and hypoxic signalling. The British journal of radiology Olcina, M. M., Kim, R. K., Melemenidis, S., Graves, E. E., Giaccia, A. J. 2018: 20180069


    The complement system is an innate immune pathway typically thought of as part of the first line of defence against "non-self" species. In the context of cancer, complement has been described to have an active role in facilitating cancer-associated processes such as increased proliferation, angiogenesis and migration. Several cellular members of the tumour microenvironment express and/or produce complement proteins locally, including tumour cells. Dysregulation of the complement system has been reported in numerous tumours and increased expression of complement activation fragments in cancer patient specimens correlates with poor patient prognosis. Importantly, genetic or pharmacological targeting of complement has been shown to reduce tumour growth in several cancer preclinical models, suggesting that complement could be an attractive therapeutic target. Hypoxia (low oxygen) is frequently found in solid tumours and has a profound biological impact on cellular and non-cellular components of the tumour microenvironment. In this review, we focus on hypoxia since this is a prevailing feature of the tumour microenvironment that, like increased complement, is typically associated with poor prognosis. Furthermore, interesting links between hypoxia and complement have been recently proposed but never collectively reviewed. Here, we explore how hypoxia alters regulation of complement proteins in different cellular components of the tumour microenvironment, as well as the downstream biological consequences of this regulation.

    View details for PubMedID 29544344

  • Macrophages Promote Circulating Tumor Cell-Mediated Local Recurrence Following Radiation Therapy in Immunosuppressed Patients. Cancer research Rafat, M. n., Aguilera, T. A., Vilalta, M. n., Bronsart, L. L., Soto, L. A., von Eyben, R. n., Golla, M. A., Ahrari, Y. n., Melemenidis, S. n., Afghahi, A. n., Jenkins, M. J., Kurian, A. W., Horst, K. n., Giaccia, A. J., Graves, E. E. 2018


    Although radiation therapy (RT) decreases the incidence of locoregional recurrence in breast cancer, patients with triple-negative breast cancer (TNBC) have increased risk of local recurrence following breast-conserving therapy (BCT). The relationship between RT and local recurrence is unknown. Here we tested the hypothesis that recurrence in some instances is due to the attraction of circulating tumor cells to irradiated tissues. To evaluate the effect of absolute lymphocyte count on local recurrence after RT in TNBC patients, we analyzed radiation effects on tumor and immune cell recruitment to tissues in an orthotopic breast cancer model. Recurrent patients exhibited a prolonged low absolute lymphocyte count when compared to non-recurrent patients following RT. Recruitment of tumor cells to irradiated normal tissues was enhanced in the absence of CD8+ T cells. Macrophages (CD11b+F480+) preceded tumor cell infiltration and were recruited to tissues following RT. Tumor cell recruitment was mitigated by inhibiting macrophage infiltration using maraviroc, an FDA-approved CCR5 receptor antagonist. Our work poses the intriguing possibility that excessive macrophage infiltration in the absence of lymphocytes promotes local recurrence after RT. This combination thus defines a high-risk group of TNBC patients.

    View details for PubMedID 29880480

  • Molecular magnetic resonance imaging of angiogenesis in vivo using polyvalent cyclic RGD-iron oxide microparticle conjugates. Theranostics Melemenidis, S., Jefferson, A., Ruparelia, N., Akhtar, A. M., Xie, J., Allen, D., Hamilton, A., Larkin, J. R., Perez-Balderas, F., Smart, S. C., Muschel, R. J., Chen, X., Sibson, N. R., Choudhury, R. P. 2015; 5 (5): 515-29


    Angiogenesis is an essential component of tumour growth and, consequently, an important target both therapeutically and diagnostically. The cell adhesion molecule α(v)β(3) integrin is a specific marker of angiogenic vessels and the most prevalent vascular integrin that binds the amino acid sequence arginine-glycine-aspartic acid (RGD). Previous studies using RGD-targeted nanoparticles (20-50 nm diameter) of iron oxide (NPIO) for magnetic resonance imaging (MRI) of tumour angiogenesis, have identified a number of limitations, including non-specific extravasation, long blood half-life (reducing specific contrast) and low targeting valency. The aim of this study, therefore, was to determine whether conjugation of a cyclic RGD variant [c(RGDyK)], with enhanced affinity for α(v)β(3), to microparticles of iron oxide (MPIO) would provide a more sensitive contrast agent for imaging of angiogenic tumour vessels. Cyclic RGD [c(RGDyK)] and RAD [c(RADyK)] based peptides were coupled to 2.8 μm MPIO, and binding efficacy tested both in vitro and in vivo. Significantly greater specific binding of c(RGDyK)-MPIO to S-nitroso-n-acetylpenicillamine (SNAP)-stimulated human umbilical vein endothelial cells in vitro than PBS-treated cells was demonstrated under both static (14-fold increase; P < 0.001) and flow (44-fold increase; P < 0.001) conditions. Subsequently, mice bearing subcutaneous colorectal (MC38) or melanoma (B16F10) derived tumours underwent in vivo MRI pre- and post-intravenous administration of c(RGDyK)-MPIO or c(RADyK)-MPIO. A significantly greater volume of MPIO-induced hypointensities were found in c(RGDyK)-MPIO injected compared to c(RADyK)-MPIO injected mice, in both tumour models (P < 0.05). Similarly, administration of c(RGDyK)-MPIO induced a greater reduction in mean tumour T(2)* relaxation times than the control agent in both tumour models (melanoma P < 0.001; colorectal P < 0.0001). Correspondingly, MPIO density per tumour volume assessed immunohistochemically was significantly greater for c(RGDyK)-MPIO than c(RADyK)-MPIO injected animals, in both melanoma (P < 0.05) and colorectal (P < 0.0005) tumours. In both cases, binding of c(RGDyK)-MPIO co-localised with α(v)β(3) expression. Comparison of RGD-targeted and dynamic contrast enhanced (DCE) MRI assessment of tumour perfusion indicated sensitivity to different vascular features. This study demonstrates specific binding of c(RGDyK)-MPIO to α(v)β(3) expressing neo-vessels, with marked and quantifiable contrast and rapid clearance of unbound particles from the blood circulation compared to NPIO. Combination of this molecular MRI approach with conventional DCE MRI will enable integrated molecular, anatomical and perfusion tumour imaging.

    View details for DOI 10.7150/thno.10319

    View details for PubMedID 25767618

    View details for PubMedCentralID PMC4350013